Fluorescence in situ hybridization (FISH) is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
When observing a three-dimensional entity through a two-dimensional projection, one dot is able to hide behind another dot. Microscope imaging projects the three-dimensional entities onto a two-dimensional sensor. When the distance between two dots is too small, separating these two overlapped dots is challenging.
The intensities of FISH dots are local maxima; therefore, image hard thresholding is believed to generate artifacts with similar intensity range. In addition, dots tend to have blurred image boundaries with relatively lower image contrast.